A cGMP phosphodiesterase was extracted from bovine rod outer segments by suspension of rod outer segments in hypotonic medium. Phosphodiesterase was further purified by chromatography on AcA 34; polyacrylamide gel electrophoresis in SGS indicated that the holoenzyme was comprised of 3 subunits (about 88, 86, and 10 kDa). Enzyme activity was markedly increased by trypsin; activity was also increased severalfold in the presence of NAD and a mono-ADP-ribosyltransferase from turkey erythrocytes. Incubation of the phosphodiesterase with the transferase and [32p]NAD indicated that both high and low molecular weight subunits were ADP-ribosylated.